The major objectives of this proposal are to gain a better understanding of insulin action in normal states and to determine those factors which are capable of directly altering insulin sensitivity in fat cells. In an attempt to better define the mechanism which underlies insulin activation of cellular effector systems, cell fusion techniques will be employed to determine if insulin binding structures in cells which fail to respond to insulin can function as true receptor. Insulin binding to adipocyte plasma membranes has recently been shown to consist of two distinct species; high affinity and low affinity binding sites. These species will be assessed in states of altered insulin sensitivity as a means of establishing the relative role of each in insulin action. Previous studies of cellular alterations in insulin resistance have been helpful in identifying cellular lesions but have not identified the systemic or cellular factors responsible for these alterations. In our laboratory we have shown that insulin and glucocorticoids can directly alter insulin sensitivity in fat tissue maintained for prolonged periods of time in a chemically-defined environment. In similar experiments we plan to investigate the influence of growth hormone, glucagon and beta-agonists (isoproterenol) on insulin action. The ability of certain substrates (carbohydrates, ketone bodies, and free fatty acids) to directly affect insulin action will also be determined. In these studies, insulin action will be evaluated in terms of insulin receptor activity, hexose transport and intracellular metabolism of glucose. The organ culture system will also be used to investigate the normalization of insulin sensitivity in adipocytes obtained from insulin resistant animals (spontaneous obese, fasted, treated with high doses of glucocorticoids). In addition, studies are proposed to determine those hormones and substrates which modulate in vitro the sensitivity of adipose tissue to glucagon and beta-agonists. The cellular action of these two hormones will be characterized by studies of hormone receptors and hormone-sensitive adenylate cyclase. Finally, the cellular alterations underlying the insulin resistance associated with uremia will be defined and it will be determined to what extent circulating factors play a role. It is anticipated that these studies will contribute to our basic knowledge of hormone action as well as to our understanding of hormone resistance in certain disease states.